US 11,773,388 B2
Target enrichment by unidirectional dual probe primer extension
Daniel Burgess, Black Earth, WI (US); Brian Christopher Godwin, Livermore, CA (US); Alexander Lovejoy, Newark, CA (US); Bronwen Miller, Cape Town (ZA); Jo-Anne Elizabeth Penkler, Cape Town (ZA); and Joseph Platzer, Oakland, CA (US)
Assigned to Roche Sequencing Solutions, Inc., Pleasanton, CA (US); and Kapa Biosystems, Inc., Wilmington, MA (US)
Filed by Roche Sequencing Solutions, Inc., Pleasanton, CA (US); and Kapa Biosystems, Inc., Wilmington, MA (US)
Filed on Jun. 22, 2020, as Appl. No. 16/907,736.
Application 16/907,736 is a continuation of application No. PCT/EP2018/085727, filed on Dec. 19, 2018.
Claims priority of provisional application 62/609,013, filed on Dec. 21, 2017.
Prior Publication US 2020/0392483 A1, Dec. 17, 2020
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/10 (2006.01); C12Q 1/6813 (2018.01); C12Q 1/6876 (2018.01); C12Q 1/6874 (2018.01)
CPC C12N 15/1034 (2013.01) [C12N 15/1072 (2013.01); C12Q 1/6813 (2013.01); C12Q 1/6874 (2013.01); C12Q 1/6876 (2013.01)] 12 Claims
OG exemplary drawing
 
1. A method for enrichment of at least one target nucleic acid in a library of nucleic acids, wherein the method comprises the following steps:
(a) hybridizing a first oligonucleotide to a target nucleic acid in the library of nucleic acids, wherein each of the nucleic acids in the library of nucleic acids has a first end comprising a first adapter and a second end comprising a second adapter;
(b) extending the hybridized first oligonucleotide with a first polymerase, thereby producing a first primer extension complex, wherein the first primer extension complex comprises the target nucleic acid and the extended first oligonucleotide;
(c) capturing the first primer extension complex;
(d) enriching the first primer extension complex relative to the library of nucleic acids;
(e) hybridizing a second oligonucleotide to the target nucleic acid;
(f) extending the hybridized second oligonucleotide with a second polymerase, thereby producing a second primer extension complex, wherein the second primer extension complex comprises the target nucleic acid and the extended second oligonucleotide, thereby liberating the extended first oligonucleotide from the first primer extension complex; and
(g) amplifying the target nucleic acid with a third polymerase, a first amplification primer, and a second amplification primer, wherein the first amplification primer has a 3′ end complementary to the first adapter and the second amplification primer has a 3′ end complementary to the second adapter, wherein the step of amplifying the target nucleic acid produces a copy of target nucleic acid, wherein the copy of the target nucleic acid comprises the entire sequence of the target nucleic acid, and wherein the copy of the target nucleic acid is of greater length than the extended first oligonucleotide and the extended second oligonucleotide.